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L3 Gene Regulation > Week 2: DNA REPLICATION - DNA Replication Machinery > Flashcards

Week 2: DNA REPLICATION - DNA Replication Machinery Flashcards

1
Q

What does semi-conservative mean ?

A

Each molecule has an original and a new strand

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2
Q

What synthesises DNA?

A

DNA polymerase

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3
Q

What phase of the cell cycle does DNA rep occur?

A

S-PHASE

8hrs

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4
Q

What does DNAp require to synthesis new DNA strand?

A

all the (A,T,G,C) deoxyribonucleoside triphosphates,
Mg2+,
3’ OH paired to DNA template

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5
Q

What direction is continuous DNA rep?

A

5->3

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6
Q

What side are DNA bases continually added?

A

3’

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7
Q

What is the byproduct of DNA polymerisation?

A

di-phosphate group

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8
Q

What is the site where DNA rep starts?

A

origin

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9
Q

How is the 3’->5’ direction replicated?

A

in fragments, discontinuous.

called Okazaki fragments

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10
Q

Draw the structure of the DNA backbone…

A

ANSWER

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11
Q

What is chromatin?

A

Highly conserved DNA

Many nucleosomes that create a chromatin fibre and eventually a chromosome

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12
Q

What makes a histone?

A

octamer

4 x dimers

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13
Q

How much DNA can an octamer of HIS wrap?

A

~1.7 turns or ~147 bases

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14
Q

What are the HIS presenting a DNA octamer?

A

H2A, H2B, H3, H4

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15
Q

What makes nucleosome?

A

An octamer of Positive HIS proteins (core)
negative backbone of DNA wraps around positive His.
“bead”

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16
Q

What alters the structure and number of nucleosomes?

A

Chromatin remodelling Enzymes

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17
Q

What can recruit chromatin remodelling enzymes?

A

Transcription activators

bind to enhancer sequences

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18
Q

What is an issue with nucleosomes and DNA rep?

A

hinderance at the origin.

machinery can’t access it

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19
Q

What is dormant origin, when are the activated?

A

activated when 2 chain are about to meet and an origin lies in the middle.

It allows no bases to be missed and chains can meet

ligase enzyme binds the 2 chains together

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20
Q

How many replication origins does EUK vs PRO have?

A 
PRO = 1 
EUK = clusters (many)
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21
Q

What 3 essential elements are needed at the replication origin?

A
  1. BS for initiator/ O rep P
  2. Start site for rep
  3. Elements for unwinding/distortion of DNA (helices) at fork
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22
Q

What are the non-essential elements for the rep O?

A

BS for activators/ chromatin remodellers

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23
Q

What is the role of the origin Rep Complex? and what does it form?

A

recruit other replication p to the origin & helicase

form pre-replactitive complex

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24
Q

what assists helicases in opening the strand?

A
  • Start site binding P
  • Cdc45 and GINS bind to MCM complex and make it active.
  • only occurs when S phase goes ahead.
  • Protect & stabilise exposed start site DNA templates.
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25
Q

What are the first things that bind to the origin of rep in EUK and PRO?

A

PRO -> initiator P
EUK -> origin recognition complex ORC

Recruit the DNA helicases to the origin region.

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26
Q

What binds to the origin of rep first?

A

ORC

origin rep complex

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27
Q

What proteins make up the helicases molecules?

active vs inactive

A

inactive =MCM hexamer- Cdt1 complex
Active= CMG helices complex, Cdc 45, MCM + GINS
2 bind and when S phase is initiated they separate to the each strand

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28
Q

How many proteins make up ORC?

A

6

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29
Q

What is the shape of the helicase?

A

ring-shape and hexameric

similar in PRO and EUK

30
Q

What is the section that separates the DNA strands?

A

NTP hydrolysis

31
Q

How does helicase move and unwrap DNA?

reaction?

A

NTP hydrolysis

change shape of protein, move rapidly along DNA strand and unwrap as it goes.

32
Q

What McM subunits bind and create a gate between with the ORC-Cdc6 complex?

A

2-5

33
Q

What element does DNAp need to bind to DNA?

A

priming group

34
Q

What is the function of the priming group?

A

provides the 3’OH group paired to DNA template required by DNA polymerases to initiate Replication

35
Q

How can priming be achieved?

A
  1. nick the DNA at origin to pose the 3’OH group
  2. pre-formed RNA acts as a primer
  3. primase enzymes synthesise RNA primers in PRO and EU
36
Q

What enzyme synthesis RNA primers?

A

primases

37
Q

What are primases?

A

small RNA polymerases
binds to NTP
Creates a short stretch or RNA with functions as the primer (chain of ribonuclease)

38
Q

Is the RNA polymerase removed?

A

YES, it is replaces with DMAp with high fidelity

transiently associated with DNA

39
Q

PRO primase?

A

DnaG

has a Zn2+ binding region for protein-protein/ protein-DNA binding

40
Q

Where does the palm on the DNAp bind to on the DNA?

A

palm binds the primer template junction and the newly synthesised DNA
through interactions with the phosphate backbone of the minor groove.

41
Q

What mediates DNTP binding?

A

by base pairing through H bonding and binding to a divalent metal cation (Mg2+)

42
Q

What is the role of the finger region of DNAp?

A

closes around the bound dNTP, prevents hydrolysis and allows nuceophillic attack of oxygen of the 3’ hydroxyl on the alpha phosphate to form a phospodiesteir bond.

43
Q

What can cause spontaneous mutation?

A

misfiring to tautness during DNA rep

occur very infrequently

44
Q

Do DNAp have proofreading activity?

A

YES!

45
Q

What is exonuclease?

A

enzymes that work by cleaving nucleotides one at a time from the end of the polynucleotide chain

46
Q

What reaction does a exonuclease undergo?

A

hydrolysis

breaks the phosphate bonds

47
Q

What are the DNAp in E.coli? PRO

A

DNA i - doesn’t replicate most of the DNA

DNAp(iii)

48
Q

how many pol3 are involved in e.coli rep?

A

3

1 for leading and 2 for Okazaki segments

49
Q

How are the RNA primers removed in PRO?

A

POL1
last step
exonucleases
and uses RNAp to synthesise gaps

50
Q

What DNAp are involved in DNA synthesis?

A

alpha
delta
epsilon

51
Q

What sub of the alpha DNAp synthesises the initiator complex? euk

A

alpha sub

3’ OH of initiator DNA is used to prime DNA synthesis of the DNAp epsilon and delta.

52
Q

What POL synthesis leading nd lagging stands?

A

leading epsilon
lagging delta
3’->5’ exonuclease proof reading activity

53
Q

What elements are RNA primers removed at the end of DNA rep? EUK

A

combined activity of DNAp and Fen1 (flap endonuclease 1)

54
Q

How does DNAp displace RNA primers? EUK

A

It extends the fragments until it displaces the RNA primers, forming a flap

Fen1 removes this flap (its an endonuclease)

55
Q

How does DNA ligase work? (general)

A

catalyses formation of a phosphodiester bond between the adjacent 3’OH and 5’P ends of the polynucleotides

H-bonded to a complementary strand

56
Q

How does DNA ligase work in EUK?

A

enzyme nucleotide intermediate

57
Q

What is adenylyl

A

.

58
Q

How to topoisomerases reduce positive supercoils?

A

formation of covalent protein-DNA intermediate allowing the DNA to rotate and re-ligate one or both strands to prevent torsional strain

59
Q

POL can only hold on to the DNA for a small amount of time (low processivity), what factors aid the prolonged binding? PRO/EUK

A

A clamp is required to increase the processivity of P

can synthesise long DNA strands

60
Q

What is the processivity of POLiii like? PRO

A

low, catalyses ~11phosdiester bonds

unbinds and binds repeatedly

61
Q

What enzyme is associated with POLiii to increase processivity?

A

HOLO enzyme

addition of many subunits

62
Q

What makes up the HOLO enzyme processive? PRO/EUK

A

many subunits but the last to attach is the clamp and makes the enzyme highly processive
catalyses thousands of phosopdiester bonds before dissociating

63
Q

What subunit is Clamp on HOLO enzyme? PRO

A

beta

64
Q

What is more processive POL1 OR POL3? E.coli PRO

A

3 as it has holo enzyme so does not dissociate from DNA as frequently
no loss of accuracy

65
Q

What is the processivity factor in EUK?

A

PCNA

66
Q

How do clamps associate with DNA?

A

the aid of clamp loader

67
Q

What are clamp loaders?

A

ATPases

68
Q

What are telomeres?

A

tandem repeats of a short conserved seq with a block of G nucleotides

69
Q

What is telomerase ?

A

essentially a reverse transcriptase

a DNA polymerase, synthesises DNA using an RNA template

70
Q

When are tolomerases present in the cell cycle?

A

on the ends of chromosome at the end of G1 phase, active during S phase

L3 Gene Regulation (14 decks)

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